Levels of 13,14-dihydro-15-keto-PGF2α in biological fluids as measured by radioimmunoassay

LEVELS

OF 1&14-DIHYDRO-l5-KETO-PGF2o AS MEASURED

Lawrence

Levine+

IN BIOLOGICAL

FLUIDS

BY RADIOIMMUNOASSAY*

and Rose Mary Gutierrez-Cernosek'

From the Graduate Brandeis University,

Department of Biochemistry Waltham, Massachusetts 02154

ABSTRACT Antibodies were prepared olite

directed

toward

in rabbits.

is immunodominant.

to a lesser extent, hydroxyl

lj,l&dihydro-15-keto-PGF20

The C-15 keto group The antibodies

the reduced

group and the C-11 hydroxyl

previously

C-15

keto group and the lj,l&double

netabolite

were

of biological cation

described

equally

samples

antibodies

of either

have been

Accepted

April

the

bond of the homologous Therefore, permits

metabolite.

metabolites

assay

identifi-

The

in human peripheral

sera and the levels of cross reacting

although the C-9

to 15-keto-PGF20

with both antisera

and quantitation

bond,

group of the metabolite.

immunodominant.

of lj,14-dihydro-15-lteto-PGF2c

recognize,

lj,l&double

With

of the metab-

levels venous in urine

determined.

25

*

This work was supported in part by a grant rrom the AmerIt is publication ican Cancer Society (Grant No. lC-LOL). JO. 852 from the Graduate De_uartment or Biochemistry, Brandeis University. .I. American Cancer Society Professor of Biochemistry (Award X0 . XP-21).

.$

Supported by Training Grant No. CA-05174 Cancer Institute, N. I. H.

JUNE

1973

VOL. 3 NO. 6

from the National

785

PROSTAGLANDINS

Radioimmunoassays for the determination of prostaglandins (PGs) of the E (l), A (2,3) and F (h-10) types have been developed.

Antibodies directed toward the two initial metabolites

of PGF2,, 15-keto-PGF2c (11) and 13,14-dihydro-15-keto-PGF2c (12), have also recently been prepared.

The latter metab-

olite is present in plasma at a concentration 20-30fold higher than the administered PGF2,, as measured by mass spectroscopy (12,13).

Except

for the report (12) of a single serum

analysis obtained by radioimmunoassay

(O-O.24 ng/ml),

levels of the 13,14-dihydro-15-keto-PGF2c

metabolite

endogenous in periph-

eral venous sera are unknown. We here report the serologic specificity of antibodies prepared in rabbits by immunization with polylysine-13,1&dihydro15-keto-PGF2c conjugates complexed to succinylated hemocyanin and their use in determining the levels of lj,l&dihydro-15keto-PGF20 in peripheral sera and in estimating PGF production in man. MATLRIALS

AND METHODS

The 13,14-dihydro-15-keto-PGF2u J. .&. Pike and U. Axen Michigan. following

The metabolite

and adjusted

methylaminopropyl) was allowed

was coupled

which

carbodiimide*HCl

in whose

synthesized.

0.005 M phosphate

NaCl.

786

were complexed

with

these antiqenic dialysis

against

of succinylated

to 2 ml with 0.14 M

2 ml of complete

received

con-

pH 7.0, 2.0 mg of the

an equal amount

were added

Each of two rabbits

We thank

overnight.

exhaustive

buffer,

and the volume was adjusted

To this mixture

adjuvant.

were added and the mixture

laboratory

After

jugates were

8

in 0.4 ml of

Eight mg of 1-ethyl-3-(3-di-

to stand at room temperature

0.15 !i NaC1, conjugate

by the

and mixed with

had been dissolved

to pH 7.2.

Dr. Helen Van Vunakis

hemocyanin

from Drs.

Kalamazoo,

to poly(L-lysine)

in 0.4 ml of dimethylformamide

mg of polylysine*HBr, water

obtained

Company,

Eight mg of 13,14-dihydro-15-keto-PGF2o

procedure.

were dissolved

was

of the Upjohn

Freund's

1 ml of this mixture

JUNE

1973

VOL.

3 NO. 6

PROSTAGLANDINS

via toe pad and intramuscular tion procedure

with

later.

The rabbits

jection

and each succeeding

week

a booster

injection,

[ 3HlPGF2a was prepared fraction

were used

were again

Boston,

for binding

with

13,14-Dihydro-15-keto-

by incubation Mass.)

of a dog lung homogenate

of [3~]~~~20, (New ting-

with

the cytoplasmic

(14).

has been reported

15-keto-[3H]PGF2a

the same as for

and the rabbits

PG metabolites

Corp.,

in-

Two weeks

week.

anti-13,14-dihydro-l5-keto-PGF2c.

land Nuclear

one week

for seven weeks.

was given

injection,

bled each succeeding Two tritiated

The same immuniza-

were bled one week after the second

after the last bleeding the primary

injection.

the same dose was repeated

The preparation

earlier

(11).

of

The ex-

tent of inhibition i3H]PGP2c

of the binding of 13,14-dihydro-15-ketoand 15-keto-[ 3H]PGF2c with anti-lj,l4-dihydro-

15-keto-PGF2c

by various

of inhibition

was the same when the

both metabolites

was

[ 3HlPGF2a prepared described

PGs was similar

Therefore,

identical.

chemically

and the sensitivity

['H]PGF2o

was used

used to prepare

the l+keto-

for most of the studies

in this report.

Radioimmunoassays

were performed

Rabbit

as follows.

antiserum diluted 1:50 in 0.01 M Tris, 0.14 M NaCl, 5.0 X -4 -4 M CaC12, pH 7.5, containing O.l,,, M MgS04, 1.5 X LO 10 gelatin,

was incubated

with

12,000 cpm) in the presence

the mixture

antibody-bound

precipitate

Bray's

PGs for

,nixture was

of goat anti-rabbit-

overnight

was collected

at approximately

was dissolved

in a modified

addition

was incubated

15-keto-L3H]PG

by centrifugation

(approximately

of unlabeled

of the reaction

After

0.3 ml or 0.4 ml.

y-globulin,

absence

The total volume

1 hr at 37O. either

L5-keto-[3H]PG or

at 2-4O.

The

as a precipitate

1,000 X g for j0 min.

The

in 0.2 ml of 0.1 N NaOH and counted

solution

in a Packard

liquid

scintillation

spectrometer.

JUNE 1973

VOL. 3 NO. 6

787

PROSTAGLANDINS

RESULTS Of the two rabbits keto-PGF2c binding

antigenic

antibodies

immunized

conjugate,

two weeks

tion and has continued

bleedings

studies

reported

precipitated

here were performed

section.

but only

This

[3~]~~~2a

homogenates

conditions,

rabbit

antiserum

that had been

or treated

benzoguinone

serum, as measured

with

reacts

The saturated

hapten,

is clearly

The

Alkaline

treatment

leads to dehydration inhibition

PGF2c3

788

to some extent group,

lOOfold;

ineffectThe C-9

since, despite

inhibition

by 15-keto-

less effective

than by 15-ketowhich probably

and loss of the C-11 hydroxyl whereas

alkaline

being

group,

treatment

its cross reactivity.

on the 13,1&-dihydro-15-keto-PGF2c

to the specificity

the most dominant

cross

role of the

of 15-keto-PGZ2,

does not affect

that four subsites

is not strikingly metabolite

from the relatively

of the 15-keto

PGE 2 is three to four times

15-keto-PGF2c

bond

(about 2$ cross reaction).

can be recognized

the immunodominance

is shown in

binding),

immunodominant

evident

by PGF2c

group

anti-

13,14-dihydro-15-keto-PGF2c,

13,1&double

ive inhibition

contribute

tissue

binding

of 15-keto-[3H]PGF2c

since the 15-keto-PGF2c

hydroxyl

creases

[3H]PGF2c

with certain

with the anti-13-l&dihydro-15-keto-

fairly extensively.

15-keto-group

PGF2,.

in the

did not bind

of the 1x,14-dihydro-15-keto-PGF2c

most effectively

immunodominant, reacts

that

2,3-dichloro-5,6-dicyano-1,4-

by inhibition

The homologous

PGF2,.

as described

incubated

(or 13,14-dihydro-15-keto-[3H]PGF2c 1.

All of the

a bleeding

(DDQ) (11).

The specificity

Fig.

hapten.

with

33%

bleeding,

4,000 cpm out of 12,000 cpm of 15-keto-[3H]PGF2c

under our radioimmunoassay Methods

In

2 kl precipitated

in a subsequent

metabolite;

injec-

The sec-

to the metabolite.

#737,

702 of the radiolabeled

2 ~1 precipitated

produced

primary

such antibodies.

antibodies

of rabbit

of the added tritiated

(8737)

one rabbit

after the initial

to produce

ond has not yet produced the earlier

with the 13,14-dihydro-15-

de-

of

It appears molecule

of anti-1x,14-dihydro-15-ketothe C-15 keto group.

JUNE 1973

Less dom-

VOL. 3 MD. 6

PROSTAGLANDINS

Rabbit

Anti-l3,14-Dihydro-15-Keto-PGF2,

13,14-Dihydro-l5-Keto-PGF

.j/ II) .x = ile

60

40

I5-Keio-PGEg

100

IO

I Nanograms

1000

Inhibitor

Fig. 1.

Inhibition of 15-keto-[%i]PGF20 rabbit anti-13,142 dihydro-15-keto-PGF2obinding. Each reaction mixture contained 0.1 ml of a I:50 dilution of rabbit antiserum, 0.1 ml of 15-

keto-[3H]PG (12,000 cpm), and 0.1 ml of unlabeled PG or PG metabolite dissolved in buffer (0.01 M Tris, 0.14 M NaCl, 5 x 10 -4 M MgSO,+, 1.5 x 10 -4 M caC12, O.l$ gelatin, pH 7.5) . Following incubation at 37O for 1 hr, 0.1 ml of goat anti-rabbit y-globulin was added and the mixtures were incubated overnight at 2-4O. The precipitate obtained from centrifugation (1000 x g for 30 min) was dissolved in 0.2 ml of 0.1 N NaOH and counted in a modified Bray's solution.

JUNE 1973

VOL. 3 NO. 6

789

inant but significant are the C-11 hydroxyl group, the C-9 hydroxyl group, and the saturated C-13,14 bond. Although the anti-13,14-dihydro-15_keto-PGF2c measures 13,14-dihydro-15-keto-PGF2o, it is not sufficiently specific to identify the inhibitor in an unknown sample.

For example, 50$ inhibition could reflect 4.0 ng of 13,14-dihydro-15-keto-

PGF2,, 7.0 ng of 15-keto-PGF2c, 35 ng of 15-keto-PGE2 or even 800 ng of PGF2,. The PGE metabolite can be eliminated from consideration by assaying the sample before and after alkaline treatment. PGF2o.

The PGF2c can be eliminated by assaying with anti-

(The latter antibodies do not cross react signifi-

cantly with the 15-keto-PGF2c metabolite.)

The contribution by 15-keto-PGF2c can be evaluated by assay with the antil+keto-PGF2o,

with which the 13,14-dihydro-15-keto-PGF2c

metabolites cross react about 5% (Fig. 2).

Therefore, with

the use of several antisera with various known specificities, =.q., anti-PGF2c, anti-15-keto-PGF2c, and anti-13,1&dihydro15-keto-PGF20, the composition of PGF and its primary metabolites can be identified and estimated. In sera and spinal fluids that we have already examined, the inhibition of the anti-13,14-dihydro-15-keto-PGF2c has been quantitatively accounted for by 13,14-dihydro-15-keto-PGF2c. Antibodies directed to both PGF2, and PGFla have previously been used to estimate levels of PGF2c in human peripheral venous sera.

In 78 samples assayed, males had more circulating PGF2,

in their blood than females, but there was a wide range in the values obtained from both males and females (males:

889 i

564 pg/ml serum: females:

For

272 z 221 pg/ml serum) (15).

these assays, the sera were treated with a methylal-ethanol system to denature the protein and release protein-bound PG into the organic phase.

After removal of the organic solvent

by evaporation, the material soluble in aqueous solvents was dialyzed against a small volume of an appropriately buffered aqueous solution and the dialysate was assayed for PGs. This procedure usually yielded 60-70~ recovery. Similar experiments

790

JUNE

1973

VOL.

3 NO. 6

PROSTAGLANDINS

Robbat

Antl-13.14-Dlhydro-l5-Keto-PGF2,

100 r

t 60

a i

Monkey

E ap

AntI-15-Keto-PGFp,

100

1 01

I

I

I

I

IO

100

Nanograms

Fig.

Inhibition

2 (top).

rabbit

are identical

(bottom). monkey

InhIbItor

of 15-keto-[3H]PGF20

anti-13,14-dihydro-l5-keto-PGF2o.

conditions

to those

Inhibition

anti-15-keto-PGF2o.

ml of a 1:50 dilution

I

1000

binding

described

in Fig.

of 15-keto-[3H]PGF20 Each reaction

of monkey

antiserum,

1.

binding

mixture

to

contained

PG or PG metab-

-4

in buffer (0.01 M Tris, 0.14 M NaCl, 5 X 10 -4 Following M CaC12, 0.176 gelatin, pH 7.5). 1.5 x 10

olite dissolved !“I

Mgs

0 4 ,

incubation globulin 2-4O.

0.1

0.1 ml of 15-keto-

(12,000 Cpm), and 0.1 ml of unlabeled

['H]PGF2d

to

The experimental

at j7O for 1 hr, 0.1 ml of rabbit

was added and the mixtures

The precipitate

obtained

for j0 min) was dissolved in a modified

JUNE 1973

Bray's

were

anti-monkey

incubated

from centrifugation

y-

overnight

at

(1000 X g

in 0.2 ml of 0.1 N NaOH and counted

solution.

VOL. 3 NO. 6

791

PROSTAGLANDINS

with 15-keto-PGF2c and 13,14-dihydro-15-keto-PGF2c also have led to 60-70s

recovery.

Using this method of extraction we

have assayed peripheral venous sera for PGF2,, 15-keto-PGF2c, and lj,l&dihydro-15-keto-PGF2c.

These levels are shown in

Fig. 3. The levels of the lj,l&dihydro-15-lceto-PGF2c metabolite are much greater than those of 15-keto-PGF2c or PGF2c in blood.

(The same serum samples were assayed with both anti-

metabolite sera (Fig. 2) to identify the metabolite being measured.) We have also measured the levels of 13,14-dihydro-15-ketocycle (Fig. 4). These values menstrual could not be correlated with ovulation (as measured by basal body temperature). Patients C and D were taking Norinyl (1+80 21-day)

PGF2, throughout the

and Gvral pills. Many of the metabolic products of PGF~~ found in urine have been identified (16,17).

Several of them (Fig. 5) have the

requisite structure (the four antigen subsites) for cross reaction with anti-13,14-dihydro-15-keto-PGF2c (the ga, lla-dihydroxy15-keto of the C-20 PG, the 7a, ga-dihydroxy-lj-keto of the C-18 and c-1.6 metabolites, and the 5a, 7a-dihydroxy-ll-keto of the C-14 metabolite).

Even some of the PGE metabolites could

cross react with this antiserum but to a lesser degree.

we

have analyzed several human urines, unextracted, for reaction The reactions

with antibodies to 13,14-dihydro-15-keto-PGF2a.

of 10 1_~1 of unextracted human urine with antisera to PGF2,, PGDl, 15-keto-PGF2a, and 13,14-dihydro-15-keto-PGF2a are shown in At the 10 ~1 level of urine, only the anti-13,14-

Table I.

dihydro-15-keto-PGF2c cross reacts significantly with metabolites present in urine. we can assign numbers for this serologic activity with anti-13,14-dihydro-15_keto-PGF2at if we assume that the c-14, c-16, and C-18 metabolites (Fig. 5) react as effectively as the C-20 metabolite. This estimate probably represents a minimal level of PGF production not Only on the basis of the above assumption but also because some PGF metabolites present do not have the requisite four subsites and therefore do not cross react.

792

JUNE 1973

VOL. 3 NO. 6

PROSTAGLANDINS

13,14-Dihydro-

I5-Kcto

I5-Kefo-PGF2,

-PGFza

PGF2.

.

IO

9

Fig. and

Levels

3.

seru-n,

represent

'-nean . The are

JUNE

of

in hulaan

DGE',,

also

1973

1~,14-dihydro-15-keto-PGF2a, sera.

the

mean

The

values,

and

the

lj,l4-dihydro-15-keto-PGF~~

15-lceto-PGFza,

expressed

standard _

as

ng/ml

deviation

levels

of

each

of

the

subject

;.ncluded.

VOL.

3 NO. 6

793

PROSTAGLANDINS

Fig. wonen

4.

pressed

794

Levels

throughout as ny/ml

of lj,14-dihydro-13-l;eto-PG1~LU the ,nenstrual cycle.

in sera of

The values

are ex-

serum.

JUNE 1973

VOL. 3 NO. 6

PROSTAGLANDINS

Fig.

5.

PGF2cz

metabolites

subjects

(16,17).

subsites

recognized

JUNE 1973

Enclosed

identified

in urine of female

areas contain

the four antigenic

by anti-13,14-dihydro-15_keto-PGF2a.

VOL. 3 NO. 6

795

PROSTAGLANDINS

The levels of PGF metabolite(s) males and 10 females

expressed

creatinine

in Table

duction

are given

of PGF/24

in the urines

of 11

both as 114124 hr and wg/g of II.

The minimal

hr would be around

value

for pro-

663-148 ILg for males and

j5-88 pg for females. We have also determined

the levels of metabolites

in rat

urines

that cross react with anti-lj,l4-dihydro-lj-keto-PGF

It was

found that in most rat urine

olites

that cross react with anti-lj,14-dihydro-15-keto-PGF and anti-PGBl

anti-15-keto-PGF2c,

All urines

(Table III).

reacted

samples

5 ~1 contained

but not with extensively

.

2a metab2aJ

anti-PGlr'2,(X

with

anti-lj,l4-

dihydro-15-keto-PGF2a

at the 10 1~1 level, but at tllis Level,

there was significant

cross reaction

with unextracted Table

rat urine)

IV are calculations

The daily production

from this inhibition as m

expressed

with respect

excretion,

The younger

PGF than man.

with

PGF metabolite/g

of

of PGF in the rat is much

less than in man, but when to creatinine

non-specific The data in

with anti-PGF2c.

obtained

5 ~1 of rat urine and are expressed creatinine.

(possibly

as a specific

activity

the rat produces

rats produce

more

more PGP than older

rats. DISCUSSION BecauSe

of the large number

quantitative measured bodies

and qualitative

groups

with

specificity

PGF compounds

is available.

toward

15-keto-PGF2o,

analysis

groups

antibodies

on

functional will

antibodies among PGFl,,

and 13,14-dihydro-

antibodies between

as

for some of the

distinguish

13,14-dihydro-15-keto-PGF20,

we have prepared

fluids,

by these procedures

of antibodies

easily

structures,

of anti-

functional

we have prepared

that do not distinguish

and PGF 2o (14).

on a battery

different

(1.5). We also have prepared

hydro-PGF20

796

PGF2, which

related

of biological

if these different

Such a library

be simplified.

PGF2,

toward

Of course,

are immunodominant,

directed

depend

by radioimmunoassay,

the PG molecules.

of closely

analyses

to lj,l4-di13,14-dihydro-PGF2a to PGFla

JUNE 1973

(PGFl,

VOL. 3 NO. 6

PROSTAGLANDINS

reacts

more effectively

than PGF2,).

bodies

to 15-keto-PGF2c

that react much more effectively

15-keto-PGF2c here,

than 13,14-dihydro-PGF2c

antibodies

tinguish

between With

PGF2,.

to 13,14-dihydro-l5-keto-PGF2c 15-keto-PGF2c

and identify

sample.

of different different

and PGF2,.

specificities,

compounds

with

As reported

do not dis-

we can begin

all of these PG compounds

Without

anti-

and 13,14-dihydro-15-keto-

the use of all of these antisera,

to guantitate biological

We have prepared

such a catalogue some physical

must be used prior

in a

of antibodies

separation

of the

to assay with a single

antiserum. As measured

by radioimmunoassay

with

in human sera have been measured. PGF2c metabolite, 13,14-dihydro-15-keto-PGF2,_, higher

than those of the metabolite,

This conclusion leagues

had been reached

(l2,l3), who quantitated

metabolite,

reported

analysis

by Samuelsson

than the administered

13, lb-wdro-15-keto-PGF2c than endogenous levels of PGF2,.

as measured

sera were reported average greater,

PGF2,.

4.8

ng/ml

(Fig. 3),

They

were

20-

Endogenous

be expected

levels of

to be higher

The only reported

endogenous

in serum is O-240 pg/ml although

less than 0.5

ng/ml

normal

in endogenous

in one

six other

(12).

Our

is considerably

and we even have observed

in a presumably

this discrepancy

and its

by mass

of PGF2,.

level of 13,14-dihydro-15-keto-PGF2c

as high as 26 ng/ml Despite

and his col-

in plasma

by radioimmunoassay, to have

and PGF2,.

the levels of PGF2,

also would

serum,

are significantly

after administration

for this major metabolite

and

15-keto-PGF2c,

that the levels of the latter metabolite

30fold higher

value

antisera,

The levels of the

13,14-dihydro-15-keto-PGF2c,

spectrometric

several

13,14-dihydro-15-keto-PGF2c

levels of 15-keto-PGF2c,

serum

levels

a value

(Fig. 4~).

in blood

of

13,14-dihydro-15-keto-PGF2c,

the levels of 13,14-dihydro-15-keto-

PGF2,

much better

reflect

of PGF2,

PGF production

or the initial

surprising,

JUNE 1973

metabolite,

since many tissues

VOL. 3 NO. 6

than do the blood

15-keto-PGF2o.

have high

15-hydroxy

This

levels

is not

PG dehydro-

797

PROSTAGLANDINS

genase

and 15-keto-PG-A13,14

With

reductase

such high concentrations

activities

(18).

ofQ,l&dihydro-15-keto-

in sera, could the PGF2, levels previously obtained PGF2a radioimmunoassay merely reflect cross reaction of 13,14with anti-PGF20?

dihydro-15-keto-PGF20

used in our laboratory, reacts

average

the anti-PGF20

13,14-dihydro-15-keto-PGF2c

Our PGF2,

less than l$.

er (6-l?“&) .

With

however,

suggests

level of 500 pg of 15-keto-PGF2o

13,14-dihydro-15-keto-PGF2o

cross

levels are considerably

The same reasoning,

average

(about g$ of the

level) reflects,

of the 13,14-dihydro-15-keto-PGF2o

anti-15-keto-PGF2o

(cross reaction

is 5$).

data are not sufficiently to calculate

of 15-keto-PGF2c

13,14-dihydro-15-keto-PGF2c

a constant

mass spectrometric production culated

have

(19).

istration

as measured

led to the assignment daily rates were

We have determined

of [3~]~~~2.

urinary

metabolites

PGF2o.

These

tion obtained

on analyses

with antiserum

calculations with

after admin-

a minimal

produc-

urine after demonstraurine did not inhibit

(Table I).

The daily produc-

from mass spectrometric

150-330

kg in males

data of the major urinary

metabolite,

were

and 20-40 bg in females.

our minimal

daily production

PGF, determined

66-148

spectrometry cedures

798

from the serologic

activities

of urine

kg in males and 35-88 pg in females.

tive analysis

of blood

of

made from the per cent inhibi-

tion rates of PGEl and PGE2, calculated

were

of

cal-

to 13,14-dihydro-15-keto-

of unextracted

non-specifically

by

of the cross reactions

10 ~1 of unextracted

tion that this quantity immune binding

were

ratio of

in each serum.

in urine,

These

our

at the low levels

from the yield of the major metabolites

tion rate for PGF based

with

or variable

to 15-keto-PGF2,

analysis,

rates to PGE

in part,

Unfortunately,

accurate

The levels of major metabolite

great-

that our

the cross reaction

analytical

by

samples,

As in compara-

levels of PG by radioimmunoassay

(20), the levels obtained

rates of

and mass

by the immunologic

pro-

are higher.

JUNE 1973

VOL. 3 NO. 6

PROSTAGLANDINS

REFERENCES 1.

Jaffe, B. M., J. W. Smith, W. T. Newton, and C. W. Parker. Radioimmunoassay for Prostaglandins. Science 171: 494, 1971.

2.

Stylos, W. A., and B. Rivets. Preparation of Specific Antiserum to Prostaglandin A. Prostaglandins 2: 103, 1972.

3.

Zusman, R. M., B. V. Caldwell, L. Speroff, and H. R. Behrman. Radioimmunoassay of the A Prostaglandins. Prostaglandins 2: 41, 1972.

4.

Levine, L., and H. Van Vunakis. Antigenic Activity of Prostaglandins. Biochem. Biophys. Res. Commun. 41: 1171, 1970.

5.

Levine, L., R. M. Gutierrez-Cernosek, and H. Van Vunakis. Specificities of Prostaglandins Bl, F AntigenAntibody Reactions. J. Biol. Chem. 24@t:a$8F2$5971.

,0.

Caldwell, B. V., S. Burstein, W. A. Brock, and L. Speroff. Radioimmunoassay of the F Prostaglandins. J. Clin. Endocr. 33: 171, 1971.

7.

Kirton, K. T., J. C. Cornette, and K. L. Barr. Characterization of Antibody to Prostaglandin F20. Biochem. Biophys. Res. Commun. 47: 903, 1972.

8.

Orczyk, G. P., and H. B. Behrman. Ovulation Blockade by Aspirin or Indomethacin. In vivo evidence for a Role of Prostaglandin in Gonadotrosii??&retions. Prostaglandins 1: 3, 1972.

9.

Gutierrez Cernosek, R. M., L. M. Morrill, and L. Levine. Levels in Peripheral Sera of Man. Prostaglandin F Prostaglandins S? 71, 1972.

10. Yu, s. c., and G. Burke. Antigenic Activity of Prostaglandins: Specificities of Prostaglandins E , A and F2o AntigenAntibody Reactions. Prostaglandins 2: $1, 1972. 11. Levine, L., and R. M. Gutierrez-Cernosek. Preparation and Specificity of Antibodies to 15-keto-prostaglandin F20. Prostaglandins 2: 281, 1972. .. 12. Granstrom, E., and B. Samuelsson. Development and Mass Spectrometric Evaluation of a Radioimmunoassay for 9a, lladihydroxy-15-ketoprost-5-enoic Acid. FEBS Letters 26: 211, 1972. 13. Samuelsson, B. Endogenous Synthesis of Prostaglandins. Third Conference on Prostaglandins in Fertility Control, WHO Research Institutet, Stockholm, 1972, p. 189.

JUNE 1973 VOL. 3 NO. 6

799

PROSTAGLANDINS

14.

Levine, L. Antibodies to Pharmacologically Active Molecules: Specificities and Some Applications of AntiProstaglandins. Pharmacological Reviews, in press.

15.

Gutierrez-Cernosek, R. M., and L. Levine. Serological Aspects of Prostaglandins. J. Reproductive Med., in press.

16.

Granstrgm, E., and B. Samuelsson. On the Metabolism of in Female Subjects. II. Structures of ;yOs;~~;;~;;:,F,ea . J. Biol. Chem. 246: 7470, 1971. ,I Granstrom, E. on the Metabolism of Prostaglandin F in E'emaleSubjects. Structures of Two Cl4 Metabolites?a sur. J. Biochem. 25: 581, 1972.

17.

18.

Samuelsson, B., E. Granstrgm, K. Green, and M. Hamberg. Metabolism of Prostaglandins. Ann. N. Y. Acad. Sci. 180: 138, 1971.

19.

Hamberg, M. Inhibition of Prostaglandin Synthesis in Man. Biochem. Biophys. Res. Commun. 49: 720, 1972.

20.

Sanuelsson, B., Convener. Round Table Discussion. vances in Biosciences 9: 1973, in press.

800

JUNE 1973

Ad-

VOL. 3 NO. 6

PROSTAGLANDINS

Table I. Inhibition of Prostaglandin Antiprostaglandin Reaction by 10 ~1 Human Urine

sub-

ject 1 2

3 4 5 6 7 8

9 10 11 12

l3 14 l5 16 17 18

19

Anti-13,14dihydrol+ketoPGF2, (S) 21

l9 15 29 17 25 9 20 18 11

17 l9 l5 l5 l9 24

29 13 10

JUNE 1973 VOL. 3 NO. 6

Anti-l+ keto-PGF2a ($1

6 6 0 5 0 0

AntiPGF2, -@J-m 1

7 3 3 0 0

1

AntiPGBl 7

5 3 1 7 3 0

0 0 0 0 0

0 0 0

3

1

0 0 0 0

5 6 0

2

2

0

5

3

2 2

3 7 1 0 4

4

0

0

3

0

4

0

3

801

PROSTAGLANDINS

Table If.

Levels

of 13,1&Dihydro-15-Keto-Prostaglandin

Metabolites*

in Human Urine Females

Males Subject

yg/24 hr

cLg/g creatinine

Subject

kg/24 hr

clg/g creatinine

1

147

85

1

88

2

107

52

2

80

58 68

3

121

55

4

66

38

3 4

85 45

58 41

5

80

65

5

35

26

6

82

45

6

59

43

7

78 89

35 41

7 8

40 50

37 52

9 10

70 52

50 5a

-

-

8

9

86

44

10

126

64

11

148

35

49 I 12 60 " 19 103 + 29 52 I 15 Mean + S.D. *., Metabolic products that cross react with anti-lj,l&dihydrol+keto-PGF2a.

802

JUNE 1973

VOL. 3 NO. 6

PRO.STAGLANDIN.9

Table

III.

Inhibition

Prostaglandin

Anti-13,14dihydro-15keto-PGF2a

Rat

+* 8

of Prostaglandin-Anti-

Reactions

by

5 ~1 of Rat Urine

Anti-l+ keto-PGF2c

AntiPGF2cl 03 0

($1

($1

35

13

40

17

36

15

5 0

34 33

13 12

0

35

12

35

15

30

1

AntiPGBl -7K 4 5 N.D.* 5 1 5

3 6

17

3

7

9

23

1-5 11

5

5

10

28

12

0

5

11

31 16

14

5 2

11

0

13 14

16

7 6

31

11

2

3 10

15 16

30

13

2

12

17

7

5

4

12

2

*

** 1 ~1 of this rat N.D. = not determined. urine was used in the assay reaction mixture.

JUNE

1973

VOL.

3 NO. 6

803

PROSTAGLANDINS

Table

Iv.

Levels of 13,14-Dihydro-15-Keto-

Prostaglandin

Metabolites

in Urine

of Female Rats Rat

1-LgPG/g creatinine 354

122

241 520

204 224

421

250

347

139

425

271

342

203

284

804

kg PG/g creatinine

(250

7xEq

Mean +, S.D.

Rat

367 +_ 88

125 192 “_ 57

JUNE 1973

VOL. 3 NO. 6